Glycosylation analysis of two cysteine proteinase inhibitors from Atlantic salmon skin: di-O-acetylated sialic acids are the major sialic acid species on N-glycans.

We have recently identified two novel cysteine proteinase inhibitors from the skin of Atlantic salmon (Salmo salar L.), named salmon kininogen and salarin. In preliminary experiments, the proteins were found to be both N- as well as O-glycosylated. In the present study we show that both proteins carry biantennary alpha2,3-sialylated N-glycans. A very high amount of O-acetylated Neu5Ac units are present in the N-glycans, comprising about 60% di-O-acetylated species. Non-O-acetylated Neu5Ac make up less than 5% of the sialic acids in the N-glycans. A small number of Neu5Acalpha2-8Neu5Ac structures were observed in the N-glycans as well. O-glycans from both proteins were recovered by reductive beta-elimination and were identified by mass spectrometric methods as mono- and disialylated core type 1 tri- and tetrasaccharides. The method used for O-glycan isolation prevented the identification of possible O-acetylation in the O-glycan-bound sialic acids, but O-acetylation was observed in one O-glycosylated peptide isolated from trypsin digest of salarin. The chemical nature of the sialic acid modifications was further studied by liquid chromatography tandem mass spectrometry of 1,2-diamino-4,5-methylenedioxybenzene-derivatized sialic acids, revealing 7-, 8-, and 9- but no 4-O-acetylation. To our knowledge, these are the first observations of sialic acid O-acetylation in N-glycans on fish species and represent clearly the most extensive N-glycan O-acetylation described on any species.     

Victor Ginsburg's influence on my research of the role of sialic acids in biological recognition

Sialic acids are monosaccharides with relatively strong acidity which belong to the most important molecules of higher animals and also occur in some microorganisms. They are bound to complex carbohydrates and occupy prominent positions, especially in cell membranes. Their structural diversity is high and, correspondingly, the mechanisms for their biosynthesis complex. Sialic acids are involved in a great number of cell functions. Due to their cell surface location these acidic molecules shield macromolecules and cells from enzymatic and immunological attacks and thus contribute to innate immunity. In contrast to this masking role, enabling, for example, blood cells and serum glycoproteins a longer life-time, sialic acids also represent recognition sites for various physiological receptors, such as the selectins and siglecs, as well as for toxins and microorganisms and thus allow their colonization. The recognition function of sialic acids can again be masked by O-acetylation, which modifies the interaction with receptors. Many viruses use sialic acids for the infection of cells. As sialic acids play also a decisive role in tumor biology, they prove to be rather versatile molecules that modulate biological and pathological cellular events in a sensitive way. Thus, they are most prominent representatives of mediators of molecular and cellular recognition.     

Intestinal barrier function of Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis> L.) post smolts is reduced by common sea cage environments and suggested as a possible physiological welfare indicator

Background  

Fish farmed under high intensity aquaculture conditions are subjected to unnatural environments that may cause stress. Therefore awareness of how to maintain good health and welfare of farmed fish is important. For Atlantic salmon held in sea cages, water flow, dissolved oxygen (DO) levels and temperature will fluctuate over time and the fish can at times be exposed to detrimentally low DO levels and high temperatures. This experimental study investigates primary and secondary stress responses of Atlantic salmon post smolts to long-term exposure to reduced and fluctuating DO levels and high water temperatures, mimicking situations in the sea cages. Plasma cortisol levels and cortisol release to the water were assessed as indicators of the primary stress response and intestinal barrier integrity and physiological functions as indicators of secondary responses to changes in environmental conditions.     

Disease resistance, stress response and effects of triploidy in growth hormone transgenic coho salmon

Diploid and triploid coho salmon Oncorhynchus kisutch transgenic for growth hormone (GH) and control coho salmon were compared for differences in disease resistance and stress response. Resistance to the bacterial pathogen Vibrio anguillarum was not affected in transgenic fish relative to their non‐transgenic counterparts when they were infected at the fry stage, but was lower in transgenic fish when infected near smolting. Vaccination against vibriosis provided equal protection to both transgenic and non‐transgenic fish. Triploid fish showed a lower resistance to vibriosis than their diploid counterparts. Diploid transgenic fish and non‐transgenic fish appeared to show similar physiological and cellular stress responses to a heat shock. These studies provide information useful for both performance and ecological risk assessments of growth‐accelerated coho salmon.     

The human Cas1 protein: a sialic acid-specific O-acetyltransferase?

Sialic acids are important sugars at the reducing end of glycoproteins and glycolipids. They are among many other functions involved in cell-cell interactions, host-pathogen recognition and the regulation of serum half-life of glycoproteins. An important modification of sialic acids is O-acetylation, which can alter or mask the biological properties of the parent sialic acid molecule. The nature of mammalian sialate-O-acetyltransferases (EC 2.3.1.45) involved in their biosynthesis is still unknown. We have identified the human CasD1 (capsule structure1 domain containing 1) gene as a candidate to encode the elusive enzyme. The human CasD1 gene encodes a protein with a serine-glycine-asparagine-histidine hydrolase domain and a hydrophobic transmembrane domain. Expression of the Cas1 protein tagged with enhanced green fluorescent protein in mammalian and insect cells directed the protein to the medial and trans-cisternae of the Golgi. Overexpression of the Cas1 protein in combination with α-N-acetyl-neuraminide α-2,8-sialyltransferase 1 (GD3 synthase) resulted in an up to 40% increased biosynthesis of 7-O-acetylated ganglioside GD3. By quantitative real-time polymerase chain reaction, we found up to 5-fold increase in CasD1 mRNA in tumor cells overexpressing O-Ac-GD3. CasD1-specific small interfering RNA reduced O-acetylation in tumor cells. These results suggest that the human Cas1 protein is directly involved in O-acetylation of α2-8-linked sialic acids.     

Behavioral Thermoregulation by Juvenile Spring and Fall Chinook Salmon, Oncorhynchus Tshawytscha, during Smoltification

Fall chinook salmon evolved to emigrate during the summer months. The shift in the temperature preference we observed in smolting fall chinook but not spring chinook salmon may reflect a phylogenetic adaptation to summer emigration by (1) providing directional orientation as fall chinook salmon move into the marine environment, (2) maintaining optimal gill function during emigration and seawater entry, and/or (3) resetting thermoregulatory set-points to support physiological homeostasis once smolted fish enter the marine environment. Phylogenetically determined temperature adaptations and responses to thermal stress may not protect fall chinook salmon from the recent higher summer water temperatures, altered annual thermal regimes, and degraded cold water refugia that result from hydropower regulation of the Columbia and Snake rivers. The long-term survival of fall chinook salmon will likely require restoration of normal annual thermographs and rigorous changes in land use practices to protect critical thermal refugia and control maximum summer water temperatures in reservoirs.     

Impact of Fc N-glycan sialylation on IgG structure

ABSTRACT

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.     

Suppression of a sialyltransferase by antisense DNA reduces invasiveness of human colon cancer cells in vitro

Transfer of terminal alpha 2,6-linked sialic acids to N-glycans is catalyzed by beta-galactoside alpha 2,6-sialyltransferase (ST6Gal I). Expression of ST6Gal I and its products is reportedly increased in colon cancers. To investigate directly the functional effects of ST6Gal I expression, human colon cancer (HT29) cells were transfected with specific antisense DNA. ST6Gal I mRNA and protein were virtually undetectable in six strains of transfected HT29 cells. ST6Gal activity was reduced to 14% of control (P<0.005) in transfected cells. Expression of terminal alpha 2,6- and alpha 2,3-linked sialic acids, and unmasked N-acetyllactosamine oligosaccharides, respectively, was assessed using flow cytometry and fluoresceinated Sambucus nigra, Maackia amurensis and Erythrina cristagalli lectins. Results indicated a major reduction in expression of alpha 2,6-linked sialic acids and counterbalancing increase in unmasked N-acetyllactosamines in antisense DNA-transfected cells, without altered expression of alpha 2,3-linked sialic acids or ganglioside profiles. The ability of transfected cells to form colonies in soft agar and to invade extracellular matrix material (Matrigel), respectively, in vitro was reduced by approx. 98% (P<0.0001) and more than 3-fold (P<0.005) compared to parental HT29 cells. These results indicate that N-glycans bearing terminal alpha 2,6-linked sialic acids may enhance the invasive potential of colon cancer cells.     

Chemoreception in the salmon louse Lepeophtheirus salmonis: an electrophysiology approach

The search for effective and long-term solutions to the problems caused by salmon lice Lepeophtheirus salmonis (Kr?yer, 1837) has increasingly included biological/ecological mechanisms to combat infestation. One aspect of this work focuses on the host-associated stimuli that parasites use to locate and discriminate a compatible host. In this study we used electrophysiological recordings made directly from the antennule of adult lice to investigate the chemosensitivity of L. salmonis to putative chemical attractants from fish flesh, prepared by soaking whole fish tissue in seawater. There was a clear physiological response to whole fish extract (WFX) with threshold sensitivity at a dilution of 10 . When WFX was size fractionated, L. salmonis showed the greatest responses to the water-soluble fractions containing compounds between 1 and 10 kDa. The results suggest that the low molecular weight, water-soluble compounds found in salmon flesh may be important in salmon lice host choice.     

Evidence for a sialic acid salvaging pathway in lepidopteran insect cells

We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.     

Behaviour during elevated water temperatures: can physiology explain movement of juvenile Atlantic salmon to cool water?

1. Temperature governs most physiological processes in animals. Ectotherms behaviourally thermoregulate by selecting habitats with temperatures regulating their body temperature for optimal physiological functioning. However, ectotherms can experience temperature extremes forcing the organisms to seek temperature refuge. 2. Fish actively avoid potentially lethal temperatures by moving to cool-water sites created by inflowing tributaries and groundwater seeps. Juvenile Atlantic salmon (Salmo salar) of different age classes exhibit different behavioural responses to elevated temperatures (>23 °C). Yearling (1+) and 2-year-old (2+) Atlantic salmon often cease feeding, abandon territorial behaviour and swim continuously in aggregations in cool-water sites; whereas young-of-the-year (0+) fish continue defending territories and foraging. 3. This study determined whether the behavioural shift in older individuals (2+) occurred when basal metabolic rate, driven by increasing water temperature, reached the maximum metabolic rate such that anaerobic pathways were recruited to provide energy to support vital processes. Behaviour (feeding and stress responses), oxygen consumption, muscle lactate and glycogen, and circulating blood lactate and glucose concentrations were measured in wild 0+ and 2+ Atlantic salmon acclimated to water temperatures between 16 and 28 °C. 4. Results indicate that oxygen consumption of the 2+ fish increased with temperature and reached a plateau at 24 °C, a temperature that corresponded to cessation of feeding and a significant increase in muscle and blood lactate levels. By contrast, oxygen consumption in 0+ fish did not reach a plateau, feeding continued and muscle lactate did not increase, even at the highest temperatures tested (28 °C). 5. To conclude, the experiment demonstrated that the 0+ and 2+ fish had different physiological responses to the elevated water temperatures. The results suggest that wild 2+ Atlantic salmon employ behavioural responses (e.g. movement to cool-water sites) at elevated temperatures in an effort to mitigate physiological imbalances associated with an inability to support basal metabolism through aerobic metabolic processes.     

Oligosaccharide profiles of the prostate specific antigen in free and complexed forms from the prostate cancer patient serum and in seminal plasma: a glycopeptide approach

The oligosaccharide structures of prostate specific antigen (PSA) are expected to be useful in discriminating prostate cancer from benign conditions both accompanied by increased serum PSA levels. A large proportion of PSA forms a covalent complex with a glycoprotein, alpha(1)-antichymotrypsin, in human blood. In the present study, the glycan profiles of free and complexed forms of PSA from cancer patient serum and of seminal plasma PSA were compared by analyzing the glycopeptides obtained by lysylendopeptidase digestion of the electrophoretically separated PSA with mass spectrometry. The profiles of the PSA N-glycans from the free and complexed molecules were quite similar to each other and consisted of fucosylated biantennary oligosaccharides as the major class. They were mostly sialylated, and a considerable sialic acid fraction was alpha2,3-linked as determined by Streptococcus pneumoniae neuraminidase digestion of the glycopeptides. In the seminal plasma PSA, high-mannose and hybrid types of oligosaccharides were predominant, and the sialic acids attached to the latter as well as to biantennary oligosaccahrides were exclusively alpha2,6-linked because they were removed by Arthrobacter ureafaciens neuraminidase but resistant to S. pneumoniae neuraminidase. Complex-type oligosaccharides from other sources were found in the seminal plasma sample, indicating that analysis of released glycans carries a risk of being misleading. The results suggest that identification of alpha2,3-linked sialic acids on PSA potentially discriminates malignant from benign conditions, if the analysis is applied to oligosaccharides specifically attached to the N-glycosylation site of PSA in either a free or a complexed form in the serum.     

Increased alpha2,3-sialylation and hyperglycosylation of N-glycans in embryonic rat cortical neurons during camptothecin-induced apoptosis

Alterations in the glycan chains of cell surface glycoconjugates are frequently involved biological processes such as cell-cell interaction, cell migration, differentiation and development. Cultured embryonic (E18) rat cortical neurons underwent apoptosis in response to camptothecin, and lectin histochemistry showed that binding to apoptotic neurons of FITC-conjugated Maackia amurensis agglutinin (MAA), which is specific for terminal alpha2,3-sialic acid residues, increased progressively with increasing concentrations of camptothecin. Analysis of the total proteins of apoptotic neurons by SDS-PAGE, and lectin blotting using HRP-labeled MAA, revealed that the expression of terminal alpha2,3-sialic acid residues on an unknown protein with an apparent molecular mass of 25.6 kDa also increased in apoptotic neurons. NP-HPLC analysis of the total cellular N-glycans of normal and apoptotic neurons demonstrated that the expression of structurally simpler biantennary types of N-glycans fell by 49% during apoptosis whereas the more branched triantennary types of N-glycans with terminal sialic acid residues increased by up to 59%. These results suggest that increased surface expression of alpha2,3-sialic acid residues and hyperglycosylation of N-glycans is a common feature of cellular responses to changes in cell physiology such as tumorigenesis and apoptosis.     

Reduction of sialic acid O-acetylation in human colonic mucins in the adenoma-carcinoma sequence

The oligo-O-acetylation of sialic acids found in normal colonic mucins is greatly reduced in colorectal cancer. Mucins prepared from cancer tissue in adenocarcinoma showed this reduction, while normal O-acetylation was detected in resection margin and control cases and total mucin sialic acid content was significantly decreased in cancer vs control samples. A reduction of the O-acetyl transferase activity catalysing the O-acetylation reaction was also found. A series of cultured human colorectal cell lines derived from the same premalignant adenomatous line, and representative of the adenoma-carcinoma sequence were examined and revealed a depletion of oligo-O-acetylation in the original diploid premalignant line, re-expression in a further premalignant line and reduction in malignant mucinous and adenocarcinoma cell lines. Reduction of sialic acid O-acetylation appears as an early event in the process of malignant transformation in human colorectal cancer.     

Regulation of sialic acid O-acetylation in human colon mucosa

The expression of O-acetylated sialic acids in human colonic mucins is developmentally regulated, and a reduction of O-acetylation has been found to be associated with the early stages of colorectal cancer. Despite this, however, little is known about the enzymatic process of sialic acid O-acetylation in human colonic mucosa. Recently, we have reported on a human colon sialate-7(9)-O-acetyltransferase capable of incorporating acetyl groups into sialic acids at the nucleotide-sugar level [Shen et al., Biol. Chem. 383 (2002), 307-317]. In this report, we show that the CMP-N-acetyl-neuraminic acid (CMP-Neu5Ac) and acetyl-CoA (AcCoA) transporters are critical components for the O-acetylation of CMP-Neu5Ac in Golgi lumen, with specific inhibition of either transporter leading to a reduction in the formation of CMP-5-N-acetyl-9-O-acetyl-neuraminic acid (CMP-Neu5,9Ac2). Moreover, the finding that 5-N-acetyl-9-O-acetyl-neuraminic acid (Neu5,9Ac2 could be transferred from neo-synthesised CMP-Neu5,9Ac2 to endogenous glycoproteins in the same Golgi vesicles, together with the observation that asialofetuin and asialo-human colon mucin are much better acceptors for Neu5,9Ac2 than asialo-bovine submandibular gland mucin, suggests that a sialyltransferase exists that preferentially utilises CMP-Neu5,9Ac2 as the donor substrate, transferring Neu5,9Ac2 to terminal Galbeta1,3(4)R- residues.     

Involvement of sialic acid in the regulation of γ--aminobutyric acid uptake activity of γ-aminobutyric acid transporter 1

The γ-aminobutyric acid (GABA) transporters (GATs) have long been recognized for their key role in the uptake of neurotransmitters. The GAT1 belongs to the family of Na(+)- and Cl(-)-coupled transport proteins, which possess 12 putative transmembrane (TM) domains and three N-glycosylation sites on the extracellular loop between TM domains 3 and 4. Previously, we demonstrated that terminal trimming of N-glycans is important for the GABA uptake activity of GAT1. In this work, we examined the effect of deficiency, removal or oxidation of surface sialic acid residues on GABA uptake activity to investigate their role in the GABA uptake of GAT1. We found that the reduced concentration of sialic acid on N-glycans was paralleled by a decreased GABA uptake activity of GAT1 in Chinese hamster ovary (CHO) Lec3 cells (mutant defective in sialic acid biosynthesis) in comparison to CHO cells. Likewise, either enzymatic removal or chemical oxidation of terminal sialic acids using sialidase or sodium periodate, respectively, resulted in a strong reduction in GAT1 activity. Kinetic analysis revealed that deficiency, removal or oxidation of terminal sialic acids did not affect the K(m) GABA values. However, deficiency and removal of terminal sialic acids of GAT1 reduced the V(max) GABA values with a reduced apparent affinity for extracellular Na(+). Oxidation of cell surface sialic acids also strongly reduced V(max) without affecting both affinities of GAT1 for GABA and Na(+), respectively. These results demonstrated for the first time that the terminal sialic acid of N-linked oligosaccharides of GAT1 plays a crucial role in the GABA transport process.     

Sialic acids attached to O-glycans modulate voltage-gated potassium channel gating

Neuronal, cardiac, and skeletal muscle action potentials are produced and conducted through the highly regulated activity of several types of voltage-gated ion channels. Voltage-gated potassium (K(v)) channels are responsible for action potential repolarization. Glycans can be attached to glycoproteins through N- and O-linkages. Previous reports described the impact of N-glycans on voltage-gated ion channel function. Here, we show that sialic acids attached through O-linkages modulate gating of K(v)2.1, K(v)4.2, and K(v)4.3. The conductance-voltage (G-V) relationships for each isoform were shifted uniquely by a depolarizing 8-16 mV under conditions of reduced sialylation. The data indicate that sialic acids modulate K(v) channel activation through apparent electrostatic mechanisms that promote channel activity. Voltage-dependent steady-state inactivation was unaffected by changes in sialylation. N-Linked sialic acids cannot be responsible for the G-V shifts because K(v)4.2 and K(v)4.3 cannot be N-glycosylated, and immunoblot analysis confirmed K(v)2.1 is not N-glycosylated. Glycosidase gel shift analysis suggested that K(v)2.1, K(v)4.2, and K(v)4.3 were O-glycosylated and sialylated. To confirm this, azide-modified sugar residues involved specifically in O-glycan and sialic acid biosynthesis were shown to incorporate into all three K(v) channel isoforms using Cu(I)-catalyzed cycloaddition chemistry. Together, the data indicate that sialic acids attached to O-glycans uniquely modulate gating of three K(v) channel isoforms that are not N-glycosylated. These data provide the first evidence that external O-glycans, with core structures distinct from N-glycans in type and number of sugar residues, can modulate K(v) channel function and thereby contribute to changes in electrical signaling that result from regulated ion channel expression and/or O-glycosylation.